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Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-offunction effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the [...]

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN-expressing self-complementary AAV vector--a vector that leads to earlier onset of gene expression compared with standard AAV vectors--led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA., Introduction Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene and loss of encoded SMN protein (1). The [...]

Niemann-Pick disease (NPD) is caused by the loss of acid sphingomy-elinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body. This global reversal of pathology translated to normal weight gain and superior recovery of motor and cognitive functions compared to animals treated by either brain or systemic injection alone. Furthermore, animals in the combination group did not generate antibodies to hASM, demonstrating the first application of systemic-mediated tolerization to improve the efficacy of brain injections. All of the animals treated by combination therapy survived in good health to an investigator-selected 54 weeks, whereas the median lifespans of the systemic-alone, brain-alone, or untreated ASM knockout groups were 47, 48, and 34 weeks, respectively. These data demonstrate that combination therapy is a promising therapeutic modality for treating NPD and suggest a potential strategy for treating disease indications that cause both visceral and CNS pathologies. acid sphingomyelinase | adeno-associated virus | immunotolerization | lysosomal storage disease | neurodegeneraton

Tay-Sachs disease is a prototypic neurodegenerative disease. Lysosomal storage of GM2 ganglioside in Tay-Sachs and the related disorder, Sandhoff disease, is caused by deficiency of [beta]-hexosaminidase A, a heterodimeric protein. Tay-Sachs-related diseases (GM2 gangliosidoses) are incurable, but gene therapy has the potential for widespread correction of the underlying lysosomal defect by means of the secretion-recapture cellular pathway for enzymatic complementation. Sandhoff mice, lacking the [beta]-subunit of hexosaminidase, manifest many signs of classical human TaySachs disease and, with an acute course, die before 20 weeks of age. We treated Sandhoff mice by stereotaxic intracranial inoculation of recombinant adeno-associated viral vectors encoding the complementing human [beta]-hexosaminidase [alpha] and [beta] subunit genes and elements, including an HIV tat sequence, to enhance protein expression and distribution. Animals survived for >1 year with sustained, widespread, and abundant enzyme delivery in the nervous system. Onset of the disease was delayed with preservation of motor function; inflammation and GM2 ganglioside storage in the brain and spinal cord was reduced. Gene delivery of [beta]-hexosaminidase A by using adeno-associated viral vectors has realistic potential for treating the human Tay-Sachs-related diseases. adeno-associated virus | GM2 gangliosidosis | neurodegeneration

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease. axonal transport | deep cerebellar nuclei | gene therapy | lysosomal storage diseases | adeno-associated virus

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxiainducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-let on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/H1F-1[alpha]/herpes simplex virus protein VP 16 and Ad2/HIF-1[alpha]/nuclear factor-[kappa]B (NF-[kappa]B)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1[alpha]VP16 and Ad2/ HIF-1[alpha]/NF-[kappa]B mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1[alpha] protein levels. These results suggest that adenovirus-mediated expression of HIF-1[alpha]/VP16 or HIF-1[alpha]/NF-[kappa]B, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes. ischemic preconditioning: myocardial protection

Expression of angiopoietins in renal epithelial and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis. Am J Physiol Renal Physiol 287: F649-F657, 2004. First published June 15, 2004; 10.1152/ajprenal.00028.2004.--The hereditary von Hippel-Lindau (VHL) syndrome predisposes sufferers to highly vascularized tumors such as renal clear cell carcinoma (RCC) and central nervous system hemangioblastoma. In RCC4 and RCC786-0 VH[L.sup.-] ceils with VHL mutations, the protein of hypoxia-inducible factor-1[alpha] (HIF-1[alpha] is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786-0 VH[L.sup.-] cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1[alpha]. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786-0 VH[L.sup.+] than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786-0 VH[L.sup.-] cells but not RPTE cells. In RCC786-0 VH[L.sup.+] cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma. hypoxia-inducible factor-[alpha] ; von Hippel-Lindau; vascular endothelial growth factor; Tie-2 receptor

The role of exogenous .nitric oxide (.NO) in the modulation of heterologous gene expression in polarized kidney epithelial cells that were stably transduced with a cDNA encoding human wild-type cystic fibrosis membrane conductance regulator (CFTR) was assessed. The results showed that .NO impaired the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms. Furthermore, the evidence indicated the potential presence of large amounts of .NO in the lung.

A study was conducted to examine the ability of deoxyspergualin (DSG) to influence the trafficking of Delta-F508-cystic fibrosis transmembrane conductance regulatory (CFTR) to the cell membrane. Chloride channel activity was analyzed using the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium. Results indicated that the DSG treatment of human CF cells restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of ([+ or -])-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanam inium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed [Beta]-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.

A series of recombinant replication-defective adenovirus gene transfer vectors with different polyadenylation signal sequences were constructed with cystic fibrosis transmembrane conductance regulators (CTRF). The recombinant adenovirus gene vectors with built-in Ad2E4ORF6 backbones exhibited efficient gene transduction capabilities during cystic fibrosis gene therapy. The recombinant adenovirus gene vectors also corrected cyclic adenosine monophosphate-mediated Cl- secretion and fluid transport through gene transduction.

Overexpression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a chloride (Cl-) channel, arrests cell growth in the G(sub 2)/M phase and increases the cell volume of monkey kidney cells. CFTR overexpression in the G551D-CFTR mutant inhibits growth slightly, and there is an increase in the number of cells in the G(sub 2)/M phase. Decrease in the threshold of Cl- channel activity in the mutant produces growth defects. CFTR is capable of downregulating its activity.

Potent derivatives of butyrates may be effective in the cure of delta F508 or cystic fibrosis (CF) mutation-induced Chlorine ion channel defects because overexpression of mutant protein causes transfers of fractions of delta F508-cystic fibrosis transmembrane regulators (CFTR) into the plasma membrane. Incubation of delta F508-CFTR expressing cells at low temperature enhances transfer and maturation of mutant CFTR proteins.

Cystic fibrosis (CF) is the most common deadly inheritable disease among Caucasians. CF is characterized by defective salt transport in organs, particularly the lungs, sweat glands, intestines, and pancreas. The major cellular defect in CF patients involves the function and regulation of chloride channels. These channels, which are proteins embedded in the membranes of epithelial cells (which form the exterior layers of most body surfaces), allow the movement of chloride ions from the cell interior to the exterior. In normal epithelial cells, the chloride channel opens when it is phosphorylated (modified by the addition of phosphate) by a kinase (a type of enzyme). In CF cells, chloride channels are present but they cannot be activated by the kinase, and thus cannot secrete chloride ions or fluid. A major research finding in 1989 was the isolation of a gene which had a mutation in 70 percent of patients with CF. The gene codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR), but the actual function of the protein has not yet been determined. This article describes work done with the gene to allow synthesis of the protein from the gene information by means of laboratory techniques. The results show that the protein is a membrane-associated protein to which sugar molecules attach during synthesis, which is common for membrane-bound proteins. In addition, this synthesized, or recombinant, CFTR can be phosphorylated by the kinase, which regulates chloride channels (as well as other proteins). Antibodies that recognize the native protein also recognize recombinant CFTR. These results indicate that recombinant CFTR is structurally and functionally equivalent to CFTR, and this will allow CFTR function to be studied with the aim of improving diagnosis and therapy of cystic fibrosis. (Consumer Summary produced by Reliance Medical Information, Inc.)

Cystic fibrosis (CF) is the most common deadly inheritable disease affecting Caucasians. CF is characterized by defective salt transport in organs, particularly the lungs, sweat glands, intestines, and pancreas. The major cellular defect in CF patients involves the function and regulation of chloride channels. These channels, which are proteins embedded in the membranes of epithelial cells (which form the exterior layers of most body surfaces), allow the movement of chloride ions from the cell interior to the exterior. In normal epithelial cells, the chloride channel opens when it is phosphorylated (modified by the addition of phosphate) by a kinase (a type of enzyme). In CF cells, chloride channels are present but they cannot be activated by the kinase, and thus cannot secrete chloride ions or fluid. A major research finding in 1989 was the isolation of a gene which contained a mutation in 70 percent of patients with CF. The gene codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR), and work is ongoing to determine the actual function of this protein. The results described in this article show that when CFTR is added to CF cells, which have the mutated CFTR and are thereby defective in chloride transport, the defect in chloride secretion is corrected. This demonstrates a causal relationship between CFTR mutations and the actual dysfunctions associated with CF. The results also suggest that CFTR is either a chloride channel or that it regulates chloride channels, but it cannot be determined which of these two possibilities is true. The study suggests that treatment of CF by correcting the underlying defect may some day be feasible. (Consumer Summary produced by Reliance Medical Information, Inc.)

Partial correction of defective [Cl.sup.-] secretion in cystic fibrosis epithelial cells by an analog of squalamine. Am J Physiol Lung Cell Mol Physiol 281: L1164-L1172, 2001.--Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated [Cl.sup.-] transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment [Cl.sup.-] transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a [Cl.sup.-] gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell [Cl.sup.-] currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-[Cl.sup.-] solution, indicating restoration of [Cl.sup.-] secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective [Cl.sup.-] secretion. cystic fibrosis transmembrane conductance regulator; nasal potential difference; Ussing chamber; whole cell patch clamp; chloride ion